Wheat cultivar h4n12-0061

ABSTRACT

The invention relates to the wheat cultivar designated H4N12-0061. Provided by the invention are the seeds, plants and derivatives of the wheat cultivar H4N12-0061. Also provided by the invention are tissue cultures of the wheat cultivar H4N12-0061 and the plants regenerated therefrom. Still further provided by the invention are methods for producing wheat plants by crossing the wheat cultivar H4N12-0061 with itself or another wheat cultivar and plants produced by such methods.

This application claims the benefit of U.S. provisional application No.62/121,860, filed Feb. 27, 2015, which is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of wheat breeding.In particular, the invention relates to the new and distinctive wheatcultivar H4N12-0061.

2. Description of Related Art

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, better agronomic quality, resistance to herbicides, andimprovements in compositional traits.

Wheat may be classified into six different market classes. Five ofthese, including common wheat, hard red winter, hard red spring, softred winter, and white, belong to the species Triticum aestivum L., andthe sixth, durum, belongs to the species Triticum turgidum L. Wheat maybe used to produce a variety of products, including, but not limited to,grain, flour, baked goods, cereals, crackers, pasta, beverages,livestock feed, biofuel, straw, construction materials, and starches.The hard wheat classes are milled into flour used for breads, while thesoft wheat classes are milled into flour used for pastries and crackers.Wheat starch is used in the food and paper industries as laundrystarches, among other products.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the wheatcultivar H4N12-0061. The invention also relates to plants produced bygrowing the seed of the wheat cultivar H4N12-0061, as well as thederivatives of such plants. Further provided are plant parts, includingcells, plant protoplasts, plant cells of a tissue culture from whichwheat plants can be regenerated, plant calli, plant clumps, and plantcells that are intact in plants or parts of plants, such as leaves,stems, roots, root tips, anthers, pistils, seed, grain, pericarp,embryo, pollen, ovules, cotyledon, hypocotyl, spike, floret, awn, lemma,shoot, tissue, petiole, cells, and meristematic cells, and the like.

In a further aspect, the invention provides a composition comprising aseed of wheat cultivar H4N12-0061 comprised in plant seed growth media.In certain embodiments, the plant seed growth media is a soil orsynthetic cultivation medium. In specific embodiments, the growth mediummay be comprised in a container or may, for example, be soil in a field.Plant seed growth media are well known to those of skill in the art andinclude, but are in no way limited to, soil or synthetic cultivationmedium. Advantageously, plant seed growth media can provide adequatephysical support for seeds and can retain moisture and/or nutritionalcomponents. Examples of characteristics for soils that may be desirablein certain embodiments can be found, for instance, in U.S. Pat. Nos.3,932,166 and 4,707,176. Synthetic plant cultivation media are also wellknown in the art and may, in certain embodiments, comprise polymers orhydrogels. Examples of such compositions are described, for example, inU.S. Pat. No. 4,241,537.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the wheat cultivar H4N12-0061, as well as plantsregenerated therefrom, wherein the regenerated wheat plant is capable ofexpressing all the morphological and physiological characteristics of aplant grown from the wheat seed designated H4N12-0061.

Yet another aspect of the current invention is a wheat plant of thewheat cultivar H4N12-0061 further comprising a single locus conversion.In one embodiment, the wheat plant is defined as comprising the singlelocus conversion and otherwise capable of expressing all of themorphological and physiological characteristics of the wheat cultivarH4N12-0061. In particular embodiments of the invention, the single locusconversion may comprise a transgenic gene which has been introduced bygenetic transformation into the wheat cultivar H4N12-0061 or aprogenitor thereof. In still other embodiments of the invention, thesingle locus conversion may comprise a dominant or recessive allele. Thelocus conversion may confer potentially any trait upon the single locusconverted plant, including, but not limited to, herbicide resistance,insect resistance, resistance to bacterial, fungal, or viral disease,male fertility or sterility, and improved nutritional quality.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid wheat seed produced by crossing a plant of the wheatcultivar H4N12-0061 to a second wheat plant. Also included in theinvention are the F₁ hybrid wheat plants grown from the hybrid seedproduced by crossing the wheat cultivar H4N12-0061 to a second wheatplant. Still further included in the invention are the seeds of an F₁hybrid plant produced with the wheat cultivar H4N12-0061 as one parent,the second generation (F₂) hybrid wheat plant grown from the seed of theF₁ hybrid plant, and the seeds of the F₂ hybrid plant.

Still yet another aspect of the invention is a method of producing wheatseeds comprising crossing a plant of the wheat cultivar H4N12-0061 toany second wheat plant, including itself or another plant of thecultivar H4N12-0061. In particular embodiments of the invention, themethod of crossing comprises the steps of: (a) planting seeds of thewheat cultivar H4N12-0061; (b) cultivating wheat plants resulting fromsaid seeds until said plants bear flowers; (c) allowing fertilization ofthe flowers of said plants; and (d) harvesting seeds produced from saidplants.

Still yet another aspect of the invention is a method of producinghybrid wheat seeds comprising crossing the wheat cultivar H4N12-0061 toa second, distinct wheat plant that is nonisogenic to the wheat cultivarH4N12-0061. In particular embodiments of the invention, the crossingcomprises the steps of: (a) planting seeds of wheat cultivar H4N12-0061and a second, distinct wheat plant, (b) cultivating the wheat plantsgrown from the seeds until the plants bear flowers; (c) crosspollinating a flower on one of the two plants with the pollen of theother plant, and (d) harvesting the seeds resulting from the crosspollinating.

Still yet another aspect of the invention is a method for developing awheat plant in a wheat breeding program comprising: (a) obtaining awheat plant, or its parts, of the cultivar H4N12-0061; and (b) employingsaid plant or parts as a source of breeding material using plantbreeding techniques. In the method, the plant breeding techniques may beselected from the group consisting of recurrent selection, massselection, bulk selection, backcrossing, pedigree breeding, geneticmarker-assisted selection and genetic transformation. In certainembodiments of the invention, the wheat plant of cultivar H4N12-0061 maybe used as the male or female parent.

Still yet another aspect of the invention is a method of producing awheat plant derived from the wheat cultivar H4N12-0061, the methodcomprising the steps of: (a) preparing a progeny plant derived fromwheat cultivar H4N12-0061 by crossing a plant of the wheat cultivarH4N12-0061 with a second wheat plant; and (b) crossing the progeny plantwith itself or a second plant to produce a progeny plant of a subsequentgeneration which is derived from a plant of the wheat cultivarH4N12-0061. In one embodiment of the invention, the method furthercomprises: (c) crossing the progeny plant of a subsequent generationwith itself or a second plant; and (d) repeating steps (b) and (c) for,in some embodiments, at least 2, 3, 4 or more additional generations toproduce an inbred wheat plant derived from the wheat cultivarH4N12-0061. Also provided by the invention is a plant produced by thisand the other methods of the invention.

In another embodiment of the invention, the method of producing a wheatplant derived from the wheat cultivar H4N12-0061 further comprises: (a)crossing the wheat cultivar H4N12-0061-derived wheat plant with itselfor another wheat plant to yield additional wheat cultivarH4N12-0061-derived progeny wheat seed; (b) growing the progeny wheatseed of step (a) under plant growth conditions to yield additional wheatcultivar H4N12-0061-derived wheat plants; and (c) repeating the crossingand growing steps of (a) and (b) to generate further wheat cultivarH4N12-0061-derived wheat plants. In specific embodiments, steps (a) and(b) may be repeated at least 1, 2, 3, 4, or 5 or more times as desired.The invention still further provides a wheat plant produced by this andthe foregoing methods.

DETAILED DESCRIPTION OF THE INVENTION

Reference now will be made in detail to the embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, not alimitation of the invention. It will be apparent to those skilled in theart that various modifications and variations may be made in the presentinvention without departing from the scope or spirit of the invention.For instance, features illustrated or described as part of oneembodiment, can be used on another embodiment to yield a still furtherembodiment.

Thus, it is intended that the present invention covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features, and aspects ofthe present invention are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only, and is not intended as limiting the broader aspects ofthe present invention

In an embodiment, the invention is directed to wheat cultivarH4N12-0061, its seeds, plants, and hybrids. Wheat cultivar H4N12-0061 isa hard red winter type common wheat bred for fall planting in the hardred winter wheat growing regions of the United States. The primary usageof wheat cultivar H4N12-0061 will be for production of grain, but it canalso be used for production of silage harvested in the soft dough stage.Wheat cultivar H4N12-0061 was selected from the cross‘KS98W0512-2-4//HV9W99-558/KS00F5-20-3’. The breeding history of thecultivar can be summarized as follows:

SELECTION YEAR GENERATION LOCATION PLOT HARVEST CRITERIA 2006 CROSSLafayette, IN 2007 F₁ Ft Collins, CO Row Bulk plot 2008 F₂ Haven, KSPlot Random head Appropriate selections maturity and green leaf duration2009 F₃ Haven, KS Plot Random head Appropriate selections maturity andgreen leaf duration 2010 F₄ Haven, KS Headrows Bulk row Appropriatematurity, height, and vigor 2011 F₅ Haven, KS Plot Random head Vigor andtest selections weight 2012 F₆ Ft Collins, CO Headrows Head selectionsRegional yield from selected data headrow 2013 F₇ Yuma, AZ Headrows Headselections Regional yield & from selected quality data headrow 2014 F₈Yuma, AZ Headrows Bulk selected Regional yield & row quality data

A variant similar to H4N12-0061 but 10-15 cm taller occurs at afrequency of 0.2% (20 plants per 10,000). A white seed variant may occurat a frequency of up to 0.25% (25 seeds per 10,000). An awnless variantmay occur at a frequency of 0.1% (10 plants per 10,000). A bronze chaffvariant may occur at a frequency of 0.1% (10/10,000). Otherwise, thiscultivar has been uniform and stable in appearance and performanceacross several generations (F₅-F₈) and environments.

In accordance with another aspect of the invention, there is provided awheat plant having the morphological and physiological characteristicsof H4N12-0061 as presented in Table 1. Those of skill in the art willrecognize that these are typical values that may vary due to environmentand that other values that are substantially equivalent are within thescope of the invention.

TABLE 1 Phenotypic Description of Wheat Cultivar H4N12-0061CHARACTERISTIC VALUES/RATINGS 1. PLANT Coleoptile anthocyanin AbsentJuvenile plant growth Erect Plant color at boot stage Green Flag leaf atboot stage: Orientation Erect Waxy Bloom Absent Anther Color Yellow 2.STEM Internode: Form Hollow Hairiness of last internode of rachisPresent Peduncle: Form Erect Length (cm) 17  Auricle: Anthocyanin AbsentHair Absent 3. HEAD (at maturity) Density Lax Shape Tapering AwnednessAwned 4. GLUMES (at maturity) Color White Shoulder Square Shoulder widthMedium Beak shape Acuminate Beak width Medium Glume length Short Glumewidth Medium Pubescence Not present 5. SEED Shape Ovate Cheek RoundedBrush Medium Brush collar Not collared Crease width 2 Crease depth 2Color Red Texture Hard Germ size Small

In an embodiment, the invention provides a composition comprising a seedof H4N12-0061 comprised in plant seed growth media. Advantageously,plant seed growth media can provide adequate physical support for seedsand can retain moisture and/or nutritional components. In certainembodiments, the plant seed growth media is a soil or syntheticcultivation medium. Any plant seed growth media known in the art may beutilized in this embodiment and the invention is in no way limited tosoil or synthetic cultivation medium. Examples of characteristics forsoils that may be desirable in certain embodiments can be found, forinstance, in U.S. Pat. Nos. 3,932,166 and 4,707,176. Plant cultivationmedia are well known in the art and may, in certain embodiments,comprise polymers, hydrogels, or the like. Examples of such compositionsare described, for example, in U.S. Pat. No. 4,241,537. In specificembodiments, the growth medium may be comprised in a container or may,for example, be soil in a field.

In another embodiment, the invention is directed to methods forproducing a wheat plant by crossing a first parent wheat plant with asecond parent wheat plant, wherein the first or second wheat plant isthe wheat plant from the cultivar H4N12-0061. In an embodiment, thefirst and second parent wheat plants may be from the cultivar H4N12-0061(i.e., self-pollination). Any methods using the cultivar H4N12-0061 arepart of this invention: selfing, backcrosses, hybrid breeding, andcrosses to populations. Any plants produced using cultivar H4N12-0061 asa parent are within the scope of this invention. In certain embodiments,the invention is also directed to cells that, upon growth anddifferentiation, produce a cultivar having essentially all of themorphological and physiological characteristics of H4N12-0061. Thepresent invention additionally contemplates, in various embodiments, awheat plant regenerated from a tissue culture of cultivar H4N12-0061.

In some embodiments of the invention, the invention is directed to atransgenic variant of H4N12-0061. A transgenic variant of H4N12-0061 maycontain at least one transgene but could contain at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more transgenes. In another embodiment, a transgenicvariant of H4N12-0061 may contain no more than 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, or 2 transgenes. Another embodiment of theinvention involves a process for producing wheat cultivar H4N12-0061further comprising a desired trait, said process comprising introducinga transgene that confers a desired trait to a wheat plant of cultivarH4N12-0061. Methods for producing transgenic plants have been developedand are well known in the art. As part of the invention, one of ordinaryskill in the art may utilize any method of producing transgenic plantswhich is currently known or yet to be developed.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available.

In certain embodiments, the desired trait may be one or more ofherbicide tolerance or resistance, insect resistance or tolerance,disease resistance or tolerance, resistance for bacterial, viral, orfungal disease, male fertility, male sterility, decreased phytate, ormodified fatty acid or carbohydrate metabolism. The specific transgenemay be any known in the art or listed herein, including, but not limitedto a polynucleotide conferring resistance to imidazolinone, dicamba,sulfonylurea, glyphosate, glufosinate, triazine, benzonitrile,cyclohexanedione, phenoxy propionic acid, and L-phosphinothricin; apolynucleotide encoding a Bacillus thuringiensis polypeptide, apolynucleotide encoding phytase, FAD-2, FAD-3, galactinol synthase or araffinose synthetic enzyme, Fusarium, Septoria, or various viruses orbacteria.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes,coding sequences, inducible, constitutive, and tissue-specificpromoters, enhancing sequences, and signal and targeting sequences.

In some embodiments, the invention comprises a H4N12-0061 plant that hasbeen developed using both genetic engineering and traditional breedingtechniques. For example, a genetic trait may have been engineered intothe genome of a particular wheat plant may then be moved into the genomeof a H4N12-0061 plant using traditional breeding techniques that arewell known in the plant breeding arts. Likewise, a genetic trait thathas been engineered into the genome of a H4N12-0061 wheat plant may thenbe moved into the genome of another cultivar using traditional breedingtechniques that are well known in the plant breeding arts. Abackcrossing approach is commonly used to move a transgene or transgenesfrom a transformed wheat cultivar into an already developed wheatcultivar, and the resulting backcross conversion plant would thencomprise the transgene(s).

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector may comprise DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid and can be used alone or incombination with other plasmids to provide transformed wheat plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the wheat plant(s).

Expression Vectors for Wheat Transformation: Marker Genes

Expression vectors may include at least one genetic marker operablylinked to a regulatory element that allows transformed cells containingthe marker to be either recovered by negative selection, i.e.,inhibiting growth of cells that do not contain the selectable markergene, or by positive selection, i.e., screening for the product encodedby the genetic marker. Many commonly used selectable marker genes forplant transformation are well known in the transformation arts, and mayinclude, for example, genes that code for enzymes that metabolicallydetoxify a selective chemical agent, which may be an antibiotic or anherbicide, or genes that encode an altered target which is insensitiveto the inhibitor. Positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, which when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Another commonly used selectable marker gene is the hygromycinphosphotransferase gene, which confers resistance to the antibiotichygromycin.

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Other selectablemarker genes confer tolerance or resistance to herbicides such asglyphosate, glufosinate, or bromoxynil, or the like.

Other selectable marker genes for plant transformation that are not ofbacterial origin may include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvyl-shikimate-3-phosphate synthase, and plantacetolactate synthase.

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase. Invivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. More recently, a geneencoding Green Fluorescent Protein (GFP) has been utilized as a markerfor gene expression in prokaryotic and eukaryotic cells. GFP and mutantsof GFP may also be used as screenable markers.

Expression Vectors for Wheat Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter. Manytypes of promoters are well known in the transformation arts, as areother regulatory elements that can be used alone or in combination withpromoters.

As used herein, “promoter” includes reference to a region of DNAupstream of the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters may be referred to as “tissue-preferred.”Promoters that initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter under environmental control. Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in wheat. Optionally, the inducible promoter may beoperably linked to a nucleotide sequence encoding a signal sequence thatis operably linked to a gene for expression in wheat. With an induciblepromoter, the rate of transcription increases in response to an inducingagent.

Any inducible promoter may be used in the present invention. Exemplaryinducible promoters include, but are not limited to, those from the ACEIsystem, which respond to copper, and the In2 gene from maize, whichresponds to benzene-sulfonamide herbicide safeners. In an embodiment,the inducible promoter may be a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter may be an inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone.

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in wheat, or is operably linked to a nucleotidesequence encoding a signal sequence that is operably linked to a genefor expression in wheat.

Many different constitutive promoters can be utilized in the presentinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses, such as the 35S promoter from CaMVand the promoters from such genes as rice actin; ubiquitin; pEMU; MAS,and maize H3 histone. The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in wheat. Thetissue-specific promoter may be operably linked to a nucleotide sequenceencoding a signal sequence that is operably linked to a gene forexpression in wheat. Plants transformed with a gene of interest operablylinked to a tissue-specific promoter may produce the protein product ofthe transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in thepresent invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene; a leaf-specific and light-inducedpromoter, such as that from cab or rubisco; an anther-specific promoter,such as that from LAT52; a pollen-specific promoter, such as that fromZml 3; or a microspore-preferred promoter, such as that from apg.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art.

Foreign Protein Genes and Agronomic Genes

Using transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that maybe harvested in a conventional manner. A foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods.

According to an embodiment of the invention, the transgenic plantprovided for commercial production of foreign protein is, or is derivedfrom a H4N12-0061 wheat plant. In another embodiment, the biomass ofinterest is or is derived from a H4N12-0061 seed. For the relativelysmall number of transgenic plants that show higher levels of expression,a genetic map can be generated, primarily via conventional restrictionfragment length polymorphism (RFLP), polymerase chain reaction (PCR),and simple sequence repeat (SSR) analysis, which identify theapproximate chromosomal location of the integrated DNA molecule. Mapinformation concerning chromosomal location is useful for proprietaryprotection of a subject transgenic plant. If unauthorized propagation isundertaken and crosses made with other germplasm, the map of theintegration region can be compared to similar maps for suspect plants,to determine if the latter have a common parentage with the subjectplant. Map comparisons would involve hybridizations, RFLP, PCR, SSR, andsequencing, all of which are conventional techniques well known in theart.

In certain embodiments, the invention comprises transformed H4N12-0061plants that express particular agronomic genes or phenotypes ofagronomic interest. Exemplary genes implicated in this regard include,but are not limited to, those categorized below:

1. Genes that Confer Tolerance or Resistance to Pests or Disease andthat Encode:

A. Plant disease tolerance or resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseasetolerance or resistance gene (R) in the plant and the product of acorresponding avirulence (Avr) gene in the pathogen. A plant variety canbe transformed with one or more cloned resistance genes to engineerplants that are resistant to specific pathogen strains.

B. A gene conferring resistance to a pest, such as nematodes.

C. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. Moreover, DNA molecules encoding8-endotoxin genes can be purchased from American Type CultureCollection, Manassas, Va., for example, under ATCC Accession Nos. 40098,67136, 31995 and 31998.

D. A lectin. The nucleotide sequence of several Clivia miniatamannose-binding lectin genes are known in the art.

E. A vitamin-binding protein such as avidin or avidin homologues.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. For example, the nucleotide sequences of ricecysteine proteinase inhibitor, cDNA encoding tobacco proteinaseinhibitor I, and Streptomyces nitrosporeus α-amylase inhibitor are knownin the art.

G. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. For example, the baculovirus expressionof cloned juvenile hormone esterase, an inactivator of juvenile hormone,is known in the art.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, it is knownthat expression cloning yields DNA coding for insect diuretic hormonereceptor and an allostatin can be identified in Diploptera puntata.Genes encoding insect-specific, paralytic neurotoxins are also known inthe art.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide is known in the art.

J. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative, or another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule. Forexample, such enzymes include, but are not limited to, a glycolyticenzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase,a callase, a transaminase, an esterase, a hydrolase, a phosphatase, akinase, a phosphorylase, a polymerase, an elastase, a chitinase, and aglucanase, whether natural or synthetic. DNA molecules that containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. The nucleotide sequences of a cDNAencoding tobacco hookworm chitinase and parsley ubi4-2 polyubiquitingene are also known in the art.

L. A molecule that stimulates signal transduction. For example, thenucleotide sequences for mung bean calmodulin cDNA clones and a maizecalmodulin cDNA clone are known in the art.

M. A hydrophobic moment peptide. For example, peptide derivatives ofTachyplesin, which inhibit fungal plant pathogens, or syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former, or a channel blocker. Forexample, heterologous expression of a cecropin-13 lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum is known in the art.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmentaffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments is known in the art.

Q. A virus-specific antibody. For example, it is known in the art thattransgenic plants expressing recombinant antibody genes are protectedfrom virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. The cloning and characterizationof a gene that encodes a bean endopolygalacturonase-inhibiting proteinis known in the art.

S. A developmental-arrestive protein produced in nature by a plant. Forexample, it has been shown that transgenic plants expressing the barleyribosome-inactivating gene have an increased resistance to fungaldisease.

T. Genes expressing proteins with antifungal action. Fusarium headblight along with deoxynivalenol both produced by the pathogen Fusariumgraminearum (Schwabe) have caused devastating losses in wheatproduction. Genes expressing proteins with antifungal action can be usedas transgenes to prevent Fusarium head blight. Various classes ofproteins have been identified. Examples include endochitinases,exochitinases, glucanases, thionins, thaumatin-like proteins, osmotins,ribosome-inactivating proteins, flavonoids, and lactoferricin. Duringinfection with Fusarium graminearum, deoxynivalenol is produced. Thereis evidence that production of deoxynivalenol increases the virulence ofthe disease. Genes with properties for detoxification of deoxynivalenolhave been engineered for use in wheat. A synthetic peptide that competeswith deoxynivalenol has been identified. Changing the ribosomes of thehost so that they have reduced affinity for deoxynivalenol has also beenused to reduce the virulence of Fusarium graminearum. Genes used to helpreduce Fusarium head blight include, but are not limited to, Tri101(Fusarium), PDR5 (yeast), tlp-1 (oat), tlp-2 (oat), leaf tlp-1 (wheat),tlp (rice), tlp-4 (oat), endochitinase, exochitinase, glucanase(Fusarium), permatin (oat), seed hordothionin (barley), alpha-thionin(wheat), acid glucanase (alfalfa), chitinase (barley and rice), classbeta II-1,3-glucanase (barley), PR5/tlp (Arabidopsis), zeamatin (maize),type 1 RIP (barley), NPR1 (Arabidopsis), lactoferrin (mammal),oxalylCoA-decarboxylase (bacterium), IAP (baculovirus), ced-9 (C.elegans), and glucanase (rice and barley).

U. A gene, for example, the H9, H10, and H21 genes, conferringresistance to a pest, such as Hessian fly, stem soft fly, cereal leafbeetle, and/or green bug.

V. A gene conferring resistance to diseases such as wheat rusts,Septoria tritici, Septoria nodorum, powdery mildew, Helminthosporiumdiseases, smuts, bunts, Fusarium diseases, bacterial diseases, and viraldiseases.

W. Genes involved in the Systemic Acquired Resistance (SAR) responseand/or the pathogenesis-related genes.

X. Antifungal genes.

Y Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone and their structurally relatedderivatives.

Z. Cystatin and cysteine proteinase inhibitors.

AA. Defensin genes.

BB. Genes conferring resistance to nematodes.

2. Genes that Confer Tolerance or Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes) andother phosphono compounds, such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus PAT bar genes), andpyridinoxy or phenoxy propionic acids and cyclohexanediones (ACCaseinhibitor-encoding genes). For example, the nucleotide sequence of aform of EPSP which can confer glyphosate resistance is known in the art.A DNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis known. Nucleotide sequences of glutamine synthetase genes that confertolerance or resistance to herbicides such as L-phosphinothricin arealso known in the art. The nucleotide sequence of a PAT gene is known inthe art, as is the production of transgenic plants that express chimericbar genes coding for PAT activity. Exemplary genes conferring resistanceto phenoxy propionic acids and cyclohexanediones, such as sethoxydim andhaloxyfop are the Accl-S1, Accl-S2, and Accl-S3 genes.

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Nucleotide sequencesfor nitrilase genes are disclosed and DNA molecules containing thesegenes are available under ATCC Accession Nos. 53435, 67441, and 67442.Cloning and expression of DNA coding for a glutathione S-transferase isdescribed in the art.

D. Acetohydroxy acid synthase. This enzyme has been found to make plantsthat express this enzyme tolerant or resistant to multiple types ofherbicides and has been introduced into a variety of plants. Other genesthat confer tolerance or resistance to herbicides include a geneencoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase, genes for glutathione reductaseand superoxide dismutase, and genes for various phosphotransferases.

E. Protoporphyrinogen oxidase (protox). Protox is necessary for theproduction of chlorophyll, which is necessary for survival in allplants. The protox enzyme serves as the target for a variety ofherbicidal compounds. These herbicides also inhibit growth of differentspecies of plants present, causing their total destruction. Thedevelopment of plants containing altered protox activity that aretolerant or resistant to these herbicides is described in the art.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant.

B. Decreased phytate content. This can be accomplished by: (1)Introduction of a phytase-encoding gene that enhances breakdown ofphytate, adding more free phosphate to the transformed plant; or (2)Up-regulation of a gene that reduces phytate content. For example, thenucleotide sequence of an Aspergillus niger phytase gene has beendescribed in the art.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch, or, a gene altering thioredoxin such as NTRand/or TRX and/or a gamma zein knockout or mutant, such as cs27, TUSC27,or en27. For example, the nucleotide sequences of Streptococcus nutansfructosyltransferase gene, Bacillus subtilis levansucrase gene, andtomato invertase genes are known in the art. Transgenic plants can beproduced that express Bacillus licheniformis alpha-amylase, thatsite-direct mutagenesis of barley alpha-amylase gene, or confer maizeendosperm starch branching enzyme II or improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase.Methods of producing high oil seed by modification of starch levels(AGP) are also known. The fatty acid modification genes mentioned abovemay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

D. Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification.

E. Altering conjugated linolenic or linoleic acid content, or LEC 1,AGP, Dekl, Superall, milps, various Ipa genes such as Ipal, Ipa3, hpt,or hggt.

F. Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt) is known,as is manipulation of antioxidant levels through alteration of ahomogentisate geranyl geranyl transferase (hggt).

G. The content of high-molecular weight gluten subunits (HMS-GS).Genomic clones have been isolated for different subunits. For example,genomic clones have transformed wheat with genes that encode a modifiedHMW-GS.

H. Increased protein metabolism, zinc and iron content, for example, byregulating the NAC gene, increasing protein metabolism by regulating theGpc-B1 gene, or regulating glutenin and gliadin genes.

I. Altered essential seed amino acids. Methods of increasingaccumulation of essential amino acids in seeds, binary methods ofincreasing accumulation of essential amino acids in seeds, alteration ofamino acid compositions in seeds, methods for altering amino acidcontent of proteins, alteration of amino acid compositions in seeds, andproteins with enhanced levels of essential amino acids all are known inthe art. Other examples may include high methionine, high threonine,plant amino acid biosynthetic enzymes, increased lysine and threonine,plant tryptophan synthase beta subunit, methionine metabolic enzymes,high sulfur, increased methionine, plant amino acid biosyntheticenzymes, engineered seed protein having higher percentage of essentialamino acids, increased lysine, increasing sulfur amino acid content,synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants, increased threonine, increased lysine, CesA: cellulose synthase, hemicellulose, UDPGdH, and RGP.

4. Genes that Control Male Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility. In addition to these methods, asystem of nuclear male sterility that includes: identifying a gene whichis critical to male fertility; silencing this native gene which iscritical to male fertility; removing the native promoter from theessential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on”, resulting in the male fertility gene notbeing transcribed, is known. Fertility is restored by inducing, orturning “on”, the promoter, which in turn allows the gene that confersmale fertility to be transcribed.

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT.

B. Introduction of various stamen-specific promoters.

C. Introduction of the barnase and the barstar genes.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.Other systems that may be used include the Gin recombinase of phage Mu,the Pin recombinase of E. coli, and the R/RS system of the pSR1 plasmid.

6. Genes that Affect Abiotic Stress Resistance.

A. Genes that affect abiotic stress resistance (including but notlimited to flowering, seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,water use efficiency can be altered through alteration of malate. Inaddition, various genes, including CBF genes and transcription factors,can be effective in mitigating the negative effects of freezing, highsalinity, and drought on plants, as well as conferring other positiveeffects on plant phenotype. Abscisic acid can be altered in plants,resulting in improved plant phenotype, such as increased yield and/orincreased tolerance to abiotic stress. Cytokinin expression can bemodified resulting in plants with increased stress tolerance, such asdrought tolerance, and/or increased yield. Nitrogen utilization can beenhanced and/or nitrogen responsiveness can be altered. Ethylene can bealtered. Plant transcription factors or transcriptional regulators ofabiotic stress can also be altered.

B. Improved tolerance to water stress from drought or high salt watercondition. The HVA1 protein belongs to the group 3 LEA proteins thatinclude other members such as wheat pMA2005, cotton D-7, carrot Dc3, andrape pLEA76. These proteins are characterized by 11-mer tandem repeatsof amino acid domains which may form a probable amphophilicalpha-helical structure that presents a hydrophilic surface with ahydrophobic stripe. The barley HVA1 gene and the wheat pMA2005 gene arehighly similar at both the nucleotide level and predicted amino acidlevel. These two monocot genes are closely related to the cotton D-7gene and carrot Dc3 gene with which they share a similar structural geneorganization. There is, therefore, a correlation between LEA geneexpression or LEA protein accumulation with stress tolerance in a numberof plants. For example, in severely dehydrated wheat seedlings, theaccumulation of high levels of group 3 LEA proteins was correlated withtissue dehydration tolerance. Studies on several Indica varieties ofrice showed that the levels of group 2 LEA proteins (also known asdehydrins) and group 3 LEA proteins in roots were significantly higherin salt-tolerant varieties compared with sensitive varieties. The barleyHVA1 gene was transformed into wheat. Transformed wheat plants showedincreased tolerance to water stress.

C. Improved water stress tolerance through increased mannitol levels viathe bacterial mannitol-1-phosphate dehydrogenase gene. It is known toproduce a plant with a genetic basis for coping with water deficit byintroduction of the bacterial mannitol-1-phosphate dehydrogenase gene,mt1D, into tobacco cells via Agrobacterium-mediated transformation. Rootand leaf tissues from transgenic plants regenerated from thesetransformed tobacco cells contained up to 100 mM mannitol. Controlplants contained no detectable mannitol. To determine whether thetransgenic tobacco plants exhibited increased tolerance to waterdeficit, the growth of transgenic plants was compared to that ofuntransformed control plants in the presence of 250 mM NaCl. After 30days of exposure to 250 mM NaCl, transgenic plants had decreased weightloss and increased height relative to their untransformed counterparts.The authors concluded that the presence of mannitol in these transformedtobacco plants contributed to water deficit tolerance at the cellularlevel.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants.

Methods for Wheat Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available.

A. Agrobacterium-Mediated Transformation. One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. A. tumefaciens and A. rhizogenes are plantpathogenic soil bacteria that genetically transform plant cells. The Tiand Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. Descriptionsof Agrobacterium vector systems and methods for Agrobacterium-mediatedgene transfer are well known in the art.

B. Direct Gene Transfer. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation, wherein DNA is carried on the surface ofmicroprojectiles measuring 1 to 4 pm. The expression vector isintroduced into plant tissues with a biolistic device that acceleratesthe microprojectiles to speeds of 300 to 600 m/s, which is sufficient topenetrate plant cell walls and membranes.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Alternatively, liposome and spheroplast fusion have beenused to introduce expression vectors into plants. Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpolyL-ornithine has also been reported. Electroporation of protoplastsand whole cells and tissues has also been described. Followingtransformation of wheat target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods that are well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular wheat cultivar using theforegoing transformation techniques could be moved into another cultivarusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile, which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asmicrosatellites, and Single Nucleotide Polymorphisms (SNPs).

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile that provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forH4N12-0061.

In addition to being used for identification of wheat cultivarH4N12-0061 and plant parts and plant cells of cultivar H4N12-0061, thegenetic profile may be used to identify a wheat plant produced throughthe use of H4N12-0061 or to verify a pedigree for progeny plantsproduced through the use of H4N12-0061. The genetic marker profile isalso useful in breeding and developing backcross conversions.

In some embodiments, the present invention comprises a wheat plantcharacterized by molecular and physiological data obtained from therepresentative sample of H4N12-0061, deposited with the American TypeCulture Collection (ATCC). Provided in further embodiments of theinvention is a wheat plant formed by the combination of the H4N12-0061plant or plant cell with another wheat plant or cell and comprising thehomozygous alleles of the variety.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. PCR detection uses twooligonucleotide primers flanking the polymorphic segment of repetitiveDNA. Repeated cycles of heat denaturation of the DNA, followed byannealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties, all SSR profiles may beperformed in the same lab.

The SSR profile of wheat plant H4N12-0061 can be used to identify plantscomprising H4N12-0061 as a parent, since such plants will comprise thesame homozygous alleles as H4N12-0061. Because the wheat cultivar isessentially homozygous at all relevant loci, most loci should have onlyone type of allele present. In contrast, a genetic marker profile of anF1 progeny should be the sum of those parents, e.g., if one parent washomozygous for allele x at a particular locus, and the other parenthomozygous for allele y at that locus, then the F1 progeny will be xy(heterozygous) at that locus. Subsequent generations of progeny producedby selection and breeding are expected to be of genotype x (homozygous),y (homozygous), or xy (heterozygous) for that locus position. When theF1 plant is selfed or sibbed for successive filial generations, thelocus should be either x or y for that position.

In addition, plants and plant parts substantially benefiting from theuse of H4N12-0061 in their development, such as H4N12-0061 comprising abackcross conversion, transgene, or genetic sterility factor, may beidentified by having a molecular marker profile with a high percentidentity to H4N12-0061. In an embodiment, such a percent identity mightbe 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to H4N12-0061.

The SSR profile of H4N12-0061 also can be used to identify essentiallyderived varieties and other progeny varieties developed from the use ofH4N12-0061, as well as cells and other plant parts thereof. Progenyplants and plant parts produced using H4N12-0061 may be identified byhaving a molecular marker profile of at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 99.5% genetic contribution from H4N12-0061, as measured byeither percent identity or percent similarity. Such progeny may befurther characterized as being within a pedigree distance of H4N12-0061,such as within 1, 2, 3, 4 or 5 or fewer cross-pollinations to a wheatplant other than H4N12-0061 or a plant that has H4N12-0061 as aprogenitor. Unique molecular profiles may be identified with othermolecular tools such as SNPs and RFLPs.

While determining the SSR genetic marker profile of a plant as describedabove, several unique SSR profiles may also be identified that did notappear in either parent plant. Such unique SSR profiles may arise duringthe breeding process from recombination or mutation. A combination ofseveral unique alleles provides a means of identifying a plant variety,an F1 progeny produced from such variety, and further progeny producedfrom such variety.

Gene Conversion

When the term “wheat plant” is used in the context of the presentinvention, this also includes any gene conversions of that cultivar.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the cultivar. For example, a varietymay be backcrossed 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times to therecurrent parent. The parental wheat plant that contributes the gene forthe desired characteristic is termed the “nonrecurrent” or “donor”parent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental wheat plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent, asit is used for several rounds in the backcrossing protocol. In a typicalbackcross protocol, the original variety of interest (recurrent parent)is crossed to a second variety (nonrecurrent parent) that carries thesingle gene of interest to be transferred. The resulting progeny fromthis cross are then crossed again to the recurrent parent and theprocess is repeated until a wheat plant is obtained wherein essentiallyall of the desired morphological and physiological characteristics ofthe recurrent parent are recovered in the converted plant, in additionto the single transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent contributes to a successfulbackcrossing procedure. The goal of a backcross protocol is to alter orsubstitute a single trait or characteristic in the original variety. Toaccomplish this, a single gene of the recurrent variety is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired morphological and physiological, constitution ofthe original variety. The choice of the particular nonrecurrent parentwill depend on the purpose of the backcross. One of the major purposesis to add commercially desirable, agronomically important traits to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele may also be transferred. In this instance, it may benecessary to introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety, but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic. Examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide tolerance or resistance,resistance for bacterial, fungal, or viral disease, insect resistance ortolerance, male fertility, enhanced nutritional quality, industrialusage, yield stability and yield enhancement. These genes are generallyinherited through the nucleus.

Introduction of a New Trait or Locus into H4N12-0061

Cultivar H4N12-0061 represents a new base genetic variety into which anew locus or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Backcross Conversions of H4N12-0061

A backcross conversion of H4N12-0061 occurs when DNA sequences areintroduced through backcrossing, with H4N12-0061 utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast two or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses, or additional crosses.Molecular marker assisted breeding or selection may be utilized toreduce the number of backcrosses necessary to achieve the backcrossconversion. For example, a backcross conversion can be made in as few astwo backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes versusunlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. Desiredtraits that may be transferred through backcross conversion include, butare not limited to, sterility (nuclear and cytoplasmic), fertilityrestoration, nutritional enhancements, drought tolerance, nitrogenutilization, altered fatty acid profile, low phytate, industrialenhancements, disease resistance or tolerance (bacterial, fungal orviral), insect resistance or tolerance, and herbicide tolerance orresistance. In addition, an introgression site itself, such as an FRTsite, Lox site, or other site specific integration site, may be insertedby backcrossing and utilized for direct insertion of one or more genesof interest into a specific plant variety. A single locus may containseveral transgenes, such as a transgene for disease resistance that, inthe same expression vector, also contains a transgene for herbicidetolerance or resistance. The gene for herbicide tolerance or resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of site specific integration system allows forthe integration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selling thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Some sources suggest from one tofour or more backcrosses, but as noted above, the number of backcrossesnecessary can be reduced with the use of molecular markers. Otherfactors, such as a genetically similar donor parent, may also reduce thenumber of backcrosses necessary. Backcrossing is easiest for simplyinherited, dominant and easily recognized traits.

One process for adding or modifying a trait or locus in wheat cultivarH4N12-0061 comprises crossing H4N12-0061 plants grown from H4N12-0061seed with plants of another wheat cultivar that comprise the desiredtrait or locus, selecting F1 progeny plants that comprise the desiredtrait or locus to produce selected F1 progeny plants, crossing theselected progeny plants with the H4N12-0061 plants to produce backcrossprogeny plants, selecting for backcross progeny plants that have thedesired trait or locus and the morphological characteristics of wheatcultivar H4N12-0061 to produce selected backcross progeny plants, andbackcrossing to H4N12-0061 three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise saidtrait or locus. The modified H4N12-0061 may be further characterized ashaving essentially all of the morphological and physiologicalcharacteristics of wheat cultivar H4N12-0061 listed in Table 1, asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to H4N12-0061 as determined by SSR markers. Theabove method may be utilized with fewer backcrosses in appropriatesituations, such as when the donor parent is highly related or markersare used in the selection step. Desired nucleic acids that may be usedinclude those nucleic acids known in the art, some of which are listedherein, that will affect traits through nucleic acid expression orinhibition. Desired loci include the introgression of FRT, Lox, andother sites for site specific integration, which may also affect adesired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny wheat seed byadding a step at the end of the process that comprises crossingH4N12-0061 with the introgressed trait or locus with a different wheatplant and harvesting the resultant first generation progeny wheat seed.

A further embodiment of the invention is a back-cross conversion ofwheat cultivar H4N12-0061. A backcross conversion occurs when DNAsequences are introduced through traditional (non-transformation)breeding techniques, such as backcrossing. DNA sequences, whethernaturally occurring or transgenes, may be introduced using thesetraditional breeding techniques. Desired traits transferred through thisprocess include, but are not limited to nutritional enhancements,industrial enhancements, disease resistance or tolerance, insectresistance or tolerance, herbicide tolerance or resistance, agronomicenhancements, grain quality enhancement, waxy starch, breedingenhancements, seed production enhancements, and male sterility.Descriptions of some of the cytoplasmic male sterility genes, nuclearmale sterility genes, chemical hybridizing agents, male fertilityrestoration genes, and methods of using the aforementioned are known.Examples of genes for other traits include: Leaf rust resistance genes(Lr series such as Lr1, Lr10, Lr21, Lr22, Lr22a, Lr32, Lr37, Lr41, Lr42,and Lr43), Fusarium head blight-resistance genes (QFhs.ndsu-3B andQFhs.ndsu-2A), powdery mildew resistance genes (Pm21), common buntresistance genes (Bt-10), and wheat streak mosaic virus resistance gene(Wsm1), Russian wheat aphid resistance genes (Dn series such as Dn1,Dn2, Dn4, and Dn5), Black stem rust resistance genes (Sr38), Yellow rustresistance genes (Yr series such as Yr 1, YrSD, Yrsu, Yr17, Yr15, andYrH52), aluminum tolerance genes (Alt(BH)), dwarf genes (Rht),vernalization genes (Vrn), Hessian fly resistance genes (H9, H10, H21,and H29), grain color genes (R/r), glyphosate resistance genes (EPSPS),glufosinate genes (bar, pat) and water stress tolerance genes (Hva 1 andmt1D). The trait of interest is transferred from the donor parent to therecurrent parent, which in this case is the wheat plant disclosedherein, H4N12-0061. Single gene traits may result from either thetransfer of a dominant allele or a recessive allele. Selection ofprogeny containing the trait of interest is done by direct selection fora trait associated with a dominant allele. Selection of progeny for atrait that is transferred via a recessive allele requires growing andselfing the first backcross to determine which plants carry therecessive alleles. Recessive traits may require additional progenytesting in successive backcross generations to determine the presence ofthe gene of interest.

Using H4N12-0061 to Develop Other Wheat Varieties

Wheat varieties such as H4N12-0061 are typically developed for use inseed and grain production. However, wheat varieties such as H4N12-0061also provide a source of breeding material that may be used to developnew wheat varieties. Plant breeding techniques known in the art and usedin a wheat plant breeding program include, but are not limited to,recurrent selection, mass selection, bulk selection, mass selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Often,combinations of these techniques are used. The development of wheatvarieties in a plant breeding program requires, in general, thedevelopment and evaluation of homozygous varieties. There are manyanalytical methods available to evaluate a new variety. The oldest andmost traditional method of analysis is the observation of phenotypictraits but genotypic analysis may also be used.

Additional Breeding Methods

In an embodiment, this invention is directed to methods for producing awheat plant by crossing a first parent wheat plant with a second parentwheat plant wherein either the first or second parent wheat plant iscultivar H4N12-0061. The other parent may be any other wheat plant, suchas a wheat plant that is part of a synthetic or natural population. Anysuch methods using wheat cultivar H4N12-0061 are part of this invention:selfing, sibbing, backcrosses, mass selection, pedigree breeding, bulkselection, hybrid production, and crosses to populations. These methodsare well known in the art and some of the more commonly used breedingmethods are described below.

The following describes breeding methods that may be used with wheatcultivar H4N12-0061 in the development of further wheat plants. One suchembodiment is a method for developing a cultivar H4N12-0061 progenywheat plant in a wheat plant breeding program comprising: obtaining thewheat plant, or a part thereof, of cultivar H4N12-0061 utilizing saidplant or plant part as a source of breeding material and selecting awheat cultivar H4N12-0061 progeny plant with molecular markers in commonwith cultivar H4N12-0061 and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in the Tablesherein. Breeding steps that may be used in the wheat plant breedingprogram include pedigree breeding, backcrossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (e.g., SSRmarkers) and the making of double haploids may be utilized.

Another method involves producing a population of wheat cultivarH4N12-0061 progeny wheat plants, comprising crossing cultivar H4N12-0061with another wheat plant, thereby producing a population of wheatplants, which, on average, derive 50% of their alleles from wheatcultivar H4N12-0061. A plant of this population may be selected andrepeatedly selfed or sibbed with a wheat cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thewheat cultivar produced by this method and that has obtained at least50% of its alleles from wheat cultivar H4N12-0061.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. Thus the invention includes wheat cultivar H4N12-0061 progenywheat plants comprising a combination of at least two cultivarH4N12-0061 traits selected from the group consisting of those listed inthe Tables herein, so that said progeny wheat plant is not significantlydifferent for said traits than wheat cultivar H4N12-0061. Usingtechniques described herein, molecular markers may be used to identifysaid progeny plant as a wheat cultivar H4N12-0061 progeny plant. Meantrait values may be used to determine whether trait differences aresignificant, and the traits may be measured on plants grown under thesame environmental conditions. Once such a variety is developed itsvalue is substantial, as it is important to advance the germplasm baseas a whole in order to maintain or improve traits such as yield, diseaseresistance or tolerance, pest resistance or tolerance, and plantperformance in extreme environmental conditions.

Progeny of wheat cultivar H4N12-0061 may also be characterized throughtheir filial relationship with wheat cultivar H4N12-0061, as forexample, being within a certain number of breeding crosses of wheatcultivar H4N12-0061. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between wheat cultivar H4N12-0061 and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4 or 5 breeding crosses of wheat cultivar H4N12-0061.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asH4N12-0061 and another wheat variety having one or more desirablecharacteristics that is lacking or which complements H4N12-0061. If thetwo original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations theheterozygous condition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F1 to F2; F2 to F3; F3 to F4; F4 to F5, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. In an embodiment,the developed variety comprises homozygous alleles at about 95% or moreof its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent but at the same time retain manycomponents of the non-recurrent parent by stopping the backcrossing atan early stage and proceeding with selling and selection. For example, awheat variety may be crossed with another variety to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC1 or BC2.Progeny are selfed and selected so that the newly developed variety hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the non-recurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new wheatvarieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of wheat cultivar H4N12-0061 comprising the stepsof crossing a plant of wheat cultivar H4N12-0061 with a donor plantcomprising a desired trait, selecting an F1 progeny plant comprising thedesired trait, and backcrossing the selected F1 progeny plant to a plantof wheat cultivar H4N12-0061. This method may further comprise the stepof obtaining a molecular marker profile of wheat cultivar H4N12-0061 andusing the molecular marker profile to select for a progeny plant withthe desired trait and the molecular marker profile of H4N12-0061. In oneembodiment, the desired trait is a mutant gene or transgene present inthe donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. H4N12-0061 is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny and selfedprogeny. The selected progeny are cross pollinated with each other toform progeny for another population. This population is planted andagain superior plants are selected to cross pollinate with each other.Recurrent selection is a cyclical process and therefore can be repeatedas many times as desired. The objective of recurrent selection is toimprove the traits of a population. The improved population can then beused as a source of breeding material to obtain new varieties forcommercial or breeding use, including the production of a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self pollination, directed pollinationcould be used as part of the breeding program.

Mutation Breeding

Mutation breeding is another method of introducing new traits into wheatcultivar H4N12-0061. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation, such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (optionally from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. In addition, mutations created in other wheatplants may be used to produce a backcross conversion of wheat cultivarH4N12-0061 that comprises such mutation. Further embodiments of theinvention are the treatment of H4N12-0061 with a mutagen and the plantproduced by mutagenesis of H4N12-0061.

Breeding with Molecular Markers

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing wheat cultivar H4N12-0061. IsozymeElectrophoresis and RFLPs have been widely used to determine geneticcomposition.

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. SingleNucleotide Polymorphisms (SNPs) may also be used to identify the uniquegenetic composition of the invention and progeny varieties retainingthat unique genetic composition. Various molecular marker techniques maybe used in combination to enhance overall resolution. Wheat DNAmolecular marker linkage maps have been rapidly constructed and widelyimplemented in genetic studies.

One use of molecular markers is QTL mapping. QTL mapping is the use ofmarkers which are known to be closely linked to alleles that havemeasurable effects on a quantitative trait. Selection in the breedingprocess is based upon the accumulation of markers linked to the positiveeffecting alleles and/or the elimination of the markers linked to thenegative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a wheat plant for which wheat cultivar H4N12-0061 is a parentcan be used to produce double haploid plants. Double haploids areproduced by the doubling of a set of chromosomes (1N) from aheterozygous plant to produce a completely homozygous individual. Thiscan be advantageous because the process omits the generations of selfingneeded to obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6. Methods for obtaining haploid plants have also been disclosedin the art.

Thus, an embodiment of this invention is a process for making asubstantially homozygous H4N12-0061 progeny plant by producing orobtaining a seed from the cross of H4N12-0061 and another wheat plantand applying double haploid methods to the F1 seed or F1 plant, or toany successive filial generation. Based on studies in maize andcurrently being conducted in wheat, such methods would decrease thenumber of generations required to produce a variety with similargenetics or characteristics to H4N12-0061.

In particular, a process of making seed retaining the molecular markerprofile of wheat cultivar H4N12-0061 is contemplated, such processcomprising obtaining or producing F1 seed for which wheat cultivarH4N12-0061 is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of wheat cultivar H4N12-0061, and selecting progeny thatretain the molecular marker profile of H4N12-0061. Descriptions of otherbreeding methods that are commonly used for different traits and cropsare known.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of wheat andregeneration of plants therefrom is well known and widely published.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce wheat plants having essentially allof the morphological and physiological characteristics of wheat cultivarH4N12-0061. Means for preparing and maintaining plant tissue culture arewell known in the art. By way of example, a tissue culture comprisingorgans has been used to produce regenerated plants.

In Table 2, yield and agronomic data collected in the Central andSouthern Plains (including parts of Colorado, Kansas, Nebraska, Texas,and Oklahoma) in 2013 and 2014 for wheat cultivar H4N12-0061 arecompared to two commercially available varieties.

TABLE 2 Comparative Data for Cultivar H4N12-0061 and Selected VarietiesTan Bacterial Barley Yellow Winter Entries Compared Yield Test WeightProtein Lodging* Spot* Streak* Dwarf* Shatter* Hardiness* H4N12-006161.55 58.61 14.8 4 3 1.5 2.5 1 2.5 WINTERHAWK 61.84 59.89 13.4 3.3 2.51.5 2 2.5 2.3 Deviation −0.29 −1.28 1.35 0.75 0.50 0.00 0.50 −1.50 0.19p-value 0.01 # Obs 43 42 2 2 2 2 1 1 16 Years 2 2 1 1 2 1 1 1 2 WinPercent 53 22 50 0 0 — 0 100 25 Test Mean 58.65 58.68 14.4 3.8 3.1 1.92.5 1.7 2.7 H4N12-0061 61.55 58.61 14.8 4 3 1.5 2.5 1 2.5 WB-CEDAR 60.1558.5 14.5 1.8 3 1.3 3 1.5 2.6 Deviation 1.40 0.12 0.32 2.25 0.00 0.25−0.50 −0.50 −0.15 p-value # Obs 43 42 2 2 2 2 1 1 16 Years 2 2 1 1 2 1 11 2 Win Percent 63 55 50 0 — 0 100 100 50 Test Mean 58.65 58.68 14.4 3.83.1 1.9 2.5 1.7 2.7 *Ratings are based on a 1-9 scale with 1 being themost resistant and 9 being the most susceptible.

DEFINITIONS

In the description and tables, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

About: Refers to embodiments or values that include the standarddeviation of the mean for a given item being measured.

Allele: Any of one or more alternative forms of a gene locus, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Aphids: Aphid resistance is scored on a scale from 1 to 9; a score of 4or less indicates resistance. Varieties scored as 1 to 5 appear normaland healthy, with numbers of aphids increasing from none to up to 300per plant. A score of 7 indicates that there are 301 to 800 aphids perplant and that the plants show slight signs of infestation. A score of 9indicates severe infestation and stunted plants with severely curled andyellow leaves.

Awn: Awn is intended to mean the elongated needle-like appendages on theflower- and seed-bearing head at the top of the cereal grain plant(e.g., wheat, common wheat, rye). Awns are attached to the lemmas.Lemmas enclose the stamen and the stigma as part of the florets. Floretsare grouped in spikelets, which in turn together comprise the head.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Cell: As used herein, the term cell includes a plant cell, whetherisolated, in tissue culture, or incorporated in a plant or plant part.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Disease Resistance: As used herein, the term disease resistance ordisease resistant is defined as the ability of plants to restrict theactivities of a specified disease, such as a fungus, virus, orbacterium.

Disease Tolerance: As used herein, the term disease tolerance or diseasetolerant is defined as the ability of plants to endure a specifieddisease (such as a fungus, virus, or bacterium) or an adverseenvironmental condition and still perform and produce in spite of thisdisorder.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Embryo: The embryo is the small plant contained within a mature seed.

Emergence (EMR): The emergence score describes the ability of a seed toemerge from the soil after planting. Each genotype is given a 1 to 9score based on its percent of emergence. A score of 1 indicates anexcellent rate and percent of emergence, an intermediate score of 5indicates an average rating and a 9 score indicates a very poor rate andpercent of emergence.

Enzymes: Molecules which can act as catalysts in biological reactions.

Essentially all of the morphological and physiological characteristics:The characteristics of a plant are recovered that are otherwise presentwhen compared in the same environment, other than occasional varianttraits that might arise during backcrossing or direct introduction of atransgene.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Gene Converted (Conversion): Gene conversion or a gene converted plantrefers to plants that are developed by backcrossing, geneticengineering, or mutation, wherein essentially all of the morphologicaland physiological characteristics of a variety are recovered, inaddition to the one or more traits transferred into the variety via theback-crossing technique, genetic engineering, or mutation.

Gene Silencing: Gene silencing refers to the interruption or suppressionof the expression of a gene at the level of transcription ortranslation.

Genotype: The genetic constitution of a cell or organism.

Glume Blotch: Glume Blotch is a disease of wheat characterized by small,irregular gray to brown spots or blotches on the glumes, althoughinfections may also occur at the nodes. The disease is caused by thefungus Stagonosporum nodorum (may also be referred to as Septorianodorum). Resistance to this disease is scored on scales that reflectthe observed extent of the disease on the leaves of the plant. Ratingscales may differ but in general a low number indicates resistance andhigher number suggests different levels of susceptibility.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Head: As used herein, the term head refers to a group of spikelets atthe top of one plant stem. The term spike also refers to the head of aplant located at the top of one plant stem.

Herbicide Resistance: As used herein, the term herbicide resistance orherbicide resistant is defined as the ability of plants to survive andreproduce following exposure to a dose of herbicide that would normallybe lethal to the plant.

Herbicide Tolerance: As used herein, the term herbicide tolerance orherbicide tolerant is defined as the ability of plants to survive andreproduce after herbicide treatment.

Insect Resistance: As used herein, the term disease resistance ordisease resistant is defined as the ability of plants to restrict theactivities of a specified insect or pest.

Insect Tolerance: As used herein, the term disease tolerance or diseasetolerant is defined as the ability of plants to endure a specifiedinsect or pest and still perform and produce in spite of this disorder.

Kernel Weight: As used herein, the term kernel weight refers to theweight of individual kernels (also called seeds), often reported as theweight of one thousand kernels or “1000 Kernel Weight.”

Leaf Rust: Leaf Rust is a disease of wheat characterized by pustulesthat are circular or slightly elliptical, that usually do not coalesce,and contain masses of orange to orange-brown spores. The disease iscaused by the fungus Puccinia recondita f. sp. tritici. Infection sitesprimarily are found on the upper surfaces of leaves and leaf sheaths,and occasionally on the neck and awns. Resistance to this disease isscored on scales that reflect the observed extent of the disease on theleaves of the plant. Rating scales may differ but in general a lownumber indicates resistance and higher number suggests different levelsof susceptibility.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Locus: A locus is a position on a genomic sequence that is usually foundby a point of reference, for example, the position of a DNA sequencethat is a gene, or part of a gene or intergenic region. A locus confersone or more traits such as, for example, male sterility, herbicidetolerance or resistance, insect resistance or tolerance, diseaseresistance or tolerance, modified fatty acid metabolism, modified phyticacid metabolism, modified carbohydrate metabolism or modified proteinmetabolism. The trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the variety bybackcrossing, a natural or induced mutation, or a transgene introducedthrough genetic transformation techniques. A locus may comprise one ormore alleles integrated at a single chromosomal location.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Maturity: As used herein, the term maturity refers to the stage of plantgrowth at which the development of the kernels is complete.

Or: As used herein is meant to mean “and/or” and be interchangeabletherewith unless explicitly indicated to refer to the alternative only.

Pedigree Distance: Pedigree distance is the relationship amonggenerations based on their ancestral links as evidenced in pedigrees. Itmay be measured by the distance of the pedigree from a given startingpoint in the ancestry.

Percent Identity: Percent identity, as used herein, refers to thecomparison of the homozygous alleles of two wheat varieties. Percentidentity is determined by comparing a statistically significant numberof the homozygous alleles of two developed varieties. For example, apercent identity of 90% between wheat variety 1 and wheat variety 2means that the two varieties have the same allele at 90% of their loci.

Percent Similarity: Percent similarity as used herein refers to thecomparison of the homozygous alleles of a wheat variety such asH4N12-0061 with another plant, and if the homozygous allele ofH4N12-0061 matches at least one of the alleles from the other plant thenthey are scored as similar. Percent similarity is determined bycomparing a statistically significant number of loci and recording thenumber of loci with similar alleles as a percentage. A percentsimilarity of 90% between H4N12-0061 and another plant means thatH4N12-0061 matches at least one of the alleles of the other plant at 90%of the loci.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Plant: As used herein, the term plant includes reference to an immatureor mature whole plant, including a plant from which seed, grain, oranthers have been removed. A seed or embryo that will produce the plantis also considered to be a plant.

Plant Height (Hgt): As used herein, the term plant height is defined asthe average height in inches or centimeters of a group of plants, asmeasured from the ground level to the tip of the head, excluding awns.

Plant Parts: As used herein, the term plant parts (or reference to “awheat plant, or a part thereof”) includes, but is not limited to,protoplasts, callus, leaves, stems, roots, root tips, anthers, pistils,seed, grain, pericarp, embryo, pollen, ovules, cotyledon, hypocotyl,spike, floret, awn, lemma, shoot, tissue, petiole, cells, andmeristematic cells.

Powdery Mildew: Powdery Mildew is a disease of wheat characterized bywhite to pale gray, fuzzy or powdery colonies of mycelia, and conidia onthe upper surfaces of leaves and leaf sheaths (especially on lowerleaves), and sometimes on the spikes. The disease is caused by thefungus Erysiphe graminis f. sp. tritici. Older fungal tissue isyellowish gray. This superficial fungal material can be rubbed offeasily with the fingers. Host tissue beneath the fungal material becomeschlorotic or necrotic and, with severe infections, the leaves may die.Eventually, black spherical fruiting structures may develop in themycelia, and can be seen without magnification. Resistance to thisdisease is scored on scales that reflect the observed extent of thedisease on the leaves of the plant. Rating scales may differ but ingeneral a low number indicates resistance and higher number suggestsdifferent levels of susceptibility.

Progeny: As used herein, progeny includes an F1 wheat plant producedfrom the cross of two wheat plants where at least one plant includeswheat cultivar H4N12-0061. Progeny further includes, but is not limitedto, subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉ and F₁₀ generationalcrosses with the recurrent parental line.

Quantitative Trait Loci (QTL): Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Rhizoctonia Root Rot: Rhizoctonia Root Rot is a disease of wheatcharacterized by sharp eyespot lesions that develop on basal leafsheaths. The disease is caused by the fungus Rhizoctonia solani. Thelesion margins are dark brown with pale, straw-colored centers and themycelia often present in the centers of lesions are easily removed byrubbing. Roots can also be affected, usually becoming brown in color andreduced in number. The disease can cause stunting and a reduction in thenumber of tillers. Resistance to this disease is scored on scales thatreflect the observed extent of the disease on the leaf sheaths of theplant and on reduced vigor of the plant. Rating scales may differ but ingeneral a low number indicates resistance and higher number suggestsdifferent levels of susceptibility.

Scab or Head Blight: Scab or Head Blight a disease of wheatcharacterized by florets (especially the outer glumes) that becomeslightly darkened and oily in appearance. The disease is caused by thefungus Fusarium which has numerous species. Spores are produced that cangive the spike and shriveled, infected kernels a bright pinkish color.Spores can produce a toxin, deoxynivalenol which can be measured with achemical test. Resistance to this disease can be measured in three ways:the extent of the disease on the spikes of the plant, the percentkernels which are visibly shriveled and the amount of deoxynivalenol inthe kernels. Rating scales may differ but in general a low numberindicates resistance and higher number suggests different levels ofsusceptibility.

SDS Sedimentation: SDS sedimentation (sodium dodecyl sedimentation) testvalues are a measure of the end-use mixing and handling properties ofbread dough and their relation to bread-making quality as a result ofthe dough's gluten quality. Higher SDS sedimentation levels reflecthigher gluten quality.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Septoria Leaf Blotch or Speckled Leaf Blotch: Speckled leaf blotch is adisease of wheat, common wheat and durum wheat characterized byirregularly shaped blotches that are at first yellow and then turnreddish brown with grayish brown dry centers, caused by the rust fungusSeptoria tritici. Resistance to this disease is scored on scales thatreflect the observed extent of the disease on the leaves of the plant.Rating scales may differ but in general a low number indicatesresistance and higher number suggests different levels ofsusceptibility.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing and/or by genetictransformation to introduce a given locus that is transgenic in origin,wherein essentially all of the morphological and physiologicalcharacteristics of a wheat cultivar are recovered in addition to thecharacteristics of the locus transferred into the variety via thebackcrossing technique or by genetic transformation. It is understoodthat once introduced into any wheat plant genome, a locus that istransgenic in origin (transgene), can be introduced by backcrossing aswith any other locus.

Soil Born Mosaic Virus: Soil born mosaic virus is a disease of wheatcharacterized by mild green to yellow mosaic, yellow-green mottling,dashes, and parallel streaks, most clearly visible on the youngest leaf.Reddish streaking and necrosis at leaf tips sometimes occurs. Stuntingcan be moderate to severe, depending on the variety. The disease iscaused by a virus which is transmitted by a soilborne fungus-likeorganism, Polymyxa graminis, which makes swimming spores that infect theroots of wheat. Resistance to this disease is scored on scales thatreflect the observed extent of the disease on the young plants. Ratingscales may differ, but in general, a low number indicates resistance anda higher number suggests different levels of susceptibility.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Stem Rust: Stem Rust is a disease of wheat characterized by pustulescontaining masses of spores that are dark reddish brown, and may occuron both sides of the leaves, on the stems, and on the spikes. Thedisease is caused by the fungus Puccinia graminis f. sp. Tritici.Resistance to this disease is scored on scales that reflect the observedextent of the disease on the leaves of the plant. Rating scales maydiffer, but in general, a low number indicates resistance and a highernumber suggests different levels of susceptibility.

Stripe Rust: Stripe rust is a disease of wheat, common wheat, durumwheat, and barley characterized by elongated rows of yellow spores onthe affected parts, caused by a rust fungus, Puccinia striiformis.Resistance to this disease is scored on scales that reflect the observedextent of the disease on the leaves of the plant. Rating scales maydiffer, but in general, a low number indicates resistance and a highernumber suggests different levels of susceptibility.

Test Weight (TWT): As used herein, the term test weight is a measure ofdensity that refers to the weight in pounds of the amount of kernelscontained in a bushel unit of volume.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli, plantclumps, and plant cells that can generate tissue culture that are intactin plants or parts of plants, such as embryos, pollen, ovules, pericarp,flowers, florets, heads, spikelets, seeds, leaves, stems, roots, roottips, anthers, pistils, awns, stems, and the like.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a wheat plant by transformation.

Deposit Information

A deposit of the wheat cultivar H4N12-0061, which is disclosed hereinabove and referenced in the claims, will be made with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209. The date of deposit is ______ and the accession number forthose deposited seeds of wheat cultivar H4N12-0061 is ATCC Accession No.______. All restrictions upon the deposit have been removed, and thedeposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed is:
 1. A plant of wheat cultivar H4N12-0061, wherein asample of seed of said cultivar has been deposited under ATCC AccessionNo. ______.
 2. A plant part of the plant of claim 1, wherein the plantpart comprises at least one cell of said plant.
 3. The plant part ofclaim 2, further defined as head, awn, leaf, pollen, ovule, embryo,cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther,floret, seed, pericarp, spike, stem, and callus.
 4. A tissue cultureproduced from the plant part of claim
 3. 5. A wheat plant regeneratedfrom the tissue culture of claim
 4. 6. A seed of wheat cultivarH4N12-0061, wherein a sample of seed of said cultivar has been depositedunder ATCC Accession No. ______.
 7. A method of producing wheat seed,wherein the method comprises crossing the plant of claim 1 with itselfor a second wheat plant.
 8. The method of claim 7, wherein the method isfurther defined as comprising crossing the plant of wheat cultivarH4N12-0061 with a second, distinct wheat plant to produce an F₁ hybridwheat seed.
 9. An F₁ hybrid wheat seed produced by the method of claim8.
 10. An F₁ hybrid wheat plant produced by growing the seed of claim 9.11. A composition comprising the seed of claim 6 comprised in plant seedgrowth media, wherein a sample of seed of said cultivar has beendeposited under ATCC Accession No. ______.
 12. The composition of claim11, wherein the growth media is soil or a synthetic cultivation medium.13. A plant of wheat cultivar H4N12-0061, further comprising a singlelocus conversion, wherein a sample of seed of wheat cultivar H4N12-0061has been deposited under ATCC Accession No. ______.
 14. The plant ofclaim 13, wherein the single locus conversion comprises a transgene. 15.A seed that produces the plant of claim 13
 16. The seed of claim 15,wherein the single locus confers a trait selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, modified fatty acid metabolism,abiotic stress resistance, altered seed amino acid composition,site-specific genetic recombination, and modified carbohydratemetabolism.
 17. The seed of claim 16, wherein the single locus conferstolerance to an herbicide selected from the group consisting ofglyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxypropionic acid, L-phosphinothricin, cyclohexanone, cyclohexanedione,triazine, and benzonitrile.
 18. The seed of claim 16, wherein the singlelocus further comprises a gene encoding a Bacillus thuringiensis (Bt)endotoxin.
 19. The seed of claim 16, wherein the single locus furthercomprises a gene encoding a protein selected from the group consistingof glutenins, gliadins, phytase, fructosyltransferase, levansucrase,α-amylase, invertase and starch branching enzyme or encoding anantisense of stearyl-ACP desaturase.
 20. The seed of claim 15, whereinthe single locus conversion comprises a transgene.
 21. The method ofclaim 8, wherein the method further comprises: (a) crossing a plantgrown from said F₁ hybrid wheat seed with itself or a different wheatplant to produce a seed of a progeny plant of a subsequent generation;(b) growing a progeny plant of a subsequent generation from said seed ofa progeny plant of a subsequent generation and crossing the progenyplant of a subsequent generation with itself or a second plant toproduce a progeny plant of a further subsequent generation; and (c)repeating steps (a) and (b) using said progeny plant of a furthersubsequent generation from step (b) in place of the plant grown fromsaid F₁ hybrid wheat seed in step (a), wherein steps (a) and (b) arerepeated with sufficient inbreeding to produce an inbred wheat plantderived from the wheat cultivar H4N12-0061.
 22. A method of producing acommodity plant product comprising collecting the commodity plantproduct from the plant of claim
 1. 23. The method of claim 22, whereinthe commodity plant product is grain, flour, baked goods, cereals,pasta, beverages, livestock feed, biofuel, straw, constructionmaterials, and starches.
 24. A wheat commodity plant product produced bythe method of claim 23, wherein the commodity plant product comprises atleast one cell of wheat cultivar H4N12-0061.